Conventional processes for protein purification typically involve cell culture methods, e.g., using either mammalian or bacterial cell lines recombinantly engineered to produce the protein of interest followed by: (a) a clarification step for the removal of cells and cellular debris, e.g., using differential centrifugation and/or filtration; and (b) one or more downstream chromatography steps to separate the protein of interest from various impurities in the clarified cell culture feed.
Protein aggregates or high molecular weight protein species are one of the important impurities that need to be removed from biopharmaceutical preparations containing a product of interest, e.g., an Fc-containing protein or an antibody molecule. For example, protein aggregates and other contaminants must be removed from biopharmaceutical preparations containing a product of interest before the product can be used in diagnostic, therapeutic or other applications. This is especially important in case of therapeutic applications and for obtaining Food and Drug Administration approval.
In case of monoclonal antibodies and Fc-containing proteins, the industry standard for purification processes typically involves a purification process, which includes several steps. One of the important steps is a purification step which employs an affinity ligand called Protein A, isolated from Staphylococcus aureus, and which binds the Fc-region of antibodies. Typically, protein aggregates also bind the Protein A column, and often end up in the same elution pool as the Fc-containing protein.
Removal of protein aggregates can be challenging as often there are similarities between the physical and chemical properties of protein aggregates and the product of interest in a biopharmaceutical preparation, which generally is a monomeric molecule. In case of Fc-containing proteins, following Protein A chromatography, one or more different methods may be used downstream for the removal of protein aggregates from biopharmaceutical preparations including, for example, size exclusion chromatography, ion exchange chromatography and hydrophobic interaction chromatography.
Previously it has been reported that some of the protein aggregates can be removed during the Protein A chromatography step by using pH gradient elution. See, e.g., PCT Publication No. WO2011/028753 and Pan et al., Analytical Biochemistry 388 (2009) 273-278, incorporated by reference herein. However, as discussed in these references, the protein aggregates are eluted off of the Protein A column after or with the elution of the Fc-containing protein.